LARGE SCALE PREPARATION OF VON WILLEBRAND FACTOR BY AFFINITY CHROMATOGRAPHY

1987 ◽  
Author(s):  
J Newman ◽  
D Farb
Keyword(s):  
Factor Viii ◽  
Affinity Chromatography ◽  
Von Willebrand Factor ◽  
Large Scale ◽  
Specific Activity ◽  
Protein A ◽  
Sephadex Column ◽  
Scale Preparation ◽  
Von Willebrand ◽  
Willebrand Factor

Treatment of bleeding in von Willebrand's disease usually consists of the infusion of cryoprecipitate or plasma. DDAVP is effective in some patients. Commercial concentrates for the treatment of Hemophilia A are ineffective as a source of von Willebrand Factor (vWF) replacement in von Willibrand's disease presumedly because of the absence of higher molecular weight forms of vWF protein. A vWF concentrate obtained during the course of preparation of an affinity purified Factor VIII may provide an alternative therapeutic agent without impacting the available Factor VIII supplies. The process used for the preparation of a highly purified Factor VIII concentrate (MonoclateTM, Armour Pharmaceutical Co.) from cryoprecipitate includes an affinity chromatography step which separates vWF/Factor VIII complex from other proteins in cryoprecipitate using an anti-vWF monoclonal antibody (C. A. Fulcher & T. S. Zimmerman, 1982). Factor VIII is then dissociated from the vWF remaining on the column and is eluted immediately by 3M sodium thiocyanante (NaSCN) as a step in the regeneration of the column. Unless the NaSCN is rapidly removed from the elutriant, the vWF activity as measured by platelet agglutination is destroyed. We have taken the NaSCN eluate and processed it immediately over an in-line G-10 or G-25 Sephadex column which removes the NaSCN while the vWF is eluted with a buffered isotonic solution. Alternatively, the vWF has been precipitated from the NaSCN by ammonium sulfate or polyethylene glycol. VWF prepared by any of these methods retains platelet agglutinating activity and has a distribution of vWF multimers similar to those of vWF in normal plasma.The potency of vWF prepared from cryoprecipitate by this process is 20 units/ml and the specific activity is 20 units/mg. In several fractionations of kilogram amounts of cryoprecipitate, vWF was isolated and subjected to lyophilization and heating at 68° for 30 hours without loss of bioactivity. Drug development in progress suggest that the combined effect of affinity chromatography and exposure to NaSCN may negate the need for a heating procedure to reduce the risk of viral transmittance.

Immunoaffinity Purification of Factor VIII Complex

1987 ◽  
Vol 57 (01) ◽  
pp. 102-105 ◽  
Author(s):  
V S Hornsey ◽  
B D Griffin ◽  
D S Pepper ◽  
L R Micklem ◽  
C V Prowse
Keyword(s):  
Monoclonal Antibody ◽  
Factor Viii ◽  
Total Protein ◽  
Specific Activity ◽  
Protein A ◽  
Immunoaffinity Purification ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Murine Monoclonal Antibodies ◽  
Ristocetin Cofactor

SummaryMurine monoclonal antibodies to human von Willebrand factor (vWf) were immobilised on Sephacryl S-1000. Various solutes were screened for their ability to elute 125 I-vWf from the immobilised antibodies. The most effective solutions were then tested to determine which allowed retention of factor VIII procoagulant activity (VIII: C) and activity of vWf measured by platelet aggregation in the presence of ristocetin (Ristocetin cofactor activity R.cof.). Finally, F VIII complex was purified from both plasma and cryoprecipitate by immunoaffinity chromatography under the selected conditions. The product had a specific activity of 45 units of VIII: C per mg of protein and 60 units of R. cof. per mg representing a 4000-fold purification from plasma. The fibrinogen and fibroneetin content were each less than 4% of the total protein with vWf accounting for 60% of the total protein in the final product. Multimer analysis of the product showed a similar pattern to normal plasma and contamination by murine monoclonal antibody was less than 300 ng per mg of protein. A novel product is thus obtained containing both clinically relevant VIII :C and R. cof. in a single vial whilst using only one specific monoclonal antibody.

Factor VIII Inhibitor Antibodies with C2 Domain Specificity Are Less inhibitory to Factor VIII Complexed with von Willebrand Factor

1996 ◽  
Vol 76 (05) ◽  
pp. 749-754 ◽  
Author(s):  
Suzuki Suzuki ◽  
Morio Arai ◽  
Kagehiro Amano ◽  
Kazuhiko Kagawa ◽  
Katsuyuki Fukutake
Keyword(s):  
Complex Formation ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Domain Specificity ◽  
Factor Viii Inhibitor ◽  
C2 Domain ◽  
Recombinant Fviii ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor

SummaryIn order to clarify the potential role of von Willebrand factor (vWf) in attenuating the inactivation of factor VIII (fVIII) by those antibodies with C2 domain specificity, we investigated a panel of 14 human antibodies to fVIII. Immunoblotting analysis localized light chain (C2 domain) epitopes for four cases, heavy chain (A2 domain) epitopes in five cases, while the remaining five cases were both light and heavy chains. The inhibitor titer was considerably higher for Kogenate, a recombinant fVIII concentrate, than for Haemate P, a fVIII/vWf complex concentrate, in all inhibitor plasmas that had C2 domain specificity. In five inhibitor plasmas with A2 domain specificity and in five with both A2 and C2 domain specificities, Kogenate gave titers similar to or lower than those with Haemate P. The inhibitory effect of IgG of each inhibitor plasma was then compared with recombinant fVIII and its complex with vWf. When compared to the other 10 inhibitor IgGs, IgG concentration, which inhibited 50% of fVIII activity (IC50), was remarkably higher for the fVIII/vWf complex than for fVIII in all the inhibitor IgGs that had C2 domain reactivity. Competition of inhibitor IgG and vWf for fVIII binding was observed in an ELISA system. In 10 inhibitors that had C2 domain reactivity, the dose dependent inhibition of fVIII-vWf complex formation was observed, while, in the group of inhibitors with A2 domain specificity, there was no inhibition of the complex formation except one case. We conclude that a subset of fVIII inhibitors, those that bind to C2 domain determinants, are less inhibitory to fVIII when it is complexed with vWf that binds to overlapping region in the C2 domain.

Molecular Biology of Factor VIII/von Willebrand Factor

1978 ◽  
Vol 40 (02) ◽  
pp. 245-251 ◽  
Author(s):  
D Meyer ◽  
P A Mc Kee ◽  
L W Hoyer ◽  
T S Zimmerman ◽  
H R Gralnick
Keyword(s):  
Molecular Biology ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Von Willebrand ◽  
Willebrand Factor

Comparative Study of Intranasal, Subcutaneous and Intravenous Administration of Desamino-D-Arginine Vasopressin (DDAVP)

1986 ◽  
Vol 55 (01) ◽  
pp. 108-111 ◽  
Author(s):  
M Köhler ◽  
P Hellstern ◽  
C Miyashita ◽  
G von Blohn ◽  
E Wenzel
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Factor Xii ◽  
Additional Advantage ◽  
Mean Values ◽  
Routes Of Administration ◽  
The Mean ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Factor Antigen

SummaryThis study was performed to evaluate the influence of different routes of administration on the efficacy of DDAVP treatment. Ten healthy volunteers received DDAVP intranasally (i.n.), subcutaneously (s.c.) and intravenously (i.v.) in a randomized cross-over trial. Factor XII and high molecular weight (HMW)-kininogen levels increased only slightly after DDAVP administration. The mean increase of factor VIII: C was 3.1 (i. v.), 2.3 (s. c.), and 1.3 (i.n.) - fold over baseline. Ristocetin cofactor (von Willebrand factor antigen) increased 3.1 (2.5), 2.0 (2.3) and 1.2 (1.2) - fold over baseline mean values after i.v., s.c. and i.n. DDAVP, respectively. The half-disappearance time of factor VIII and von Willebrand factor (vWF) after DDAVP ranged from five (factor VIII: C) to eight hours (vWF). The mean increase of fibrinolytic activity was more pronounced after i.v. DDAVP. The antidiuretic effect was moderate with no apparent differences between the routes of application. This study provides further evidence that both i.v. and s.c. DDAVP administration result in an appropriate and reliable stimulation of haemostasis. An additional advantage of s. c. administration is its suitability for home treatment.

Interactions of Purified Rat Factor VIII/von Willebrand Factor with Rat and Human Platelets – Effect of Albumin and Ristocetin

1984 ◽  
Vol 52 (01) ◽  
pp. 057-059 ◽  
Author(s):  
E Dejana ◽  
M Furlan ◽  
B Barbieri ◽  
M B Donati ◽  
E A Beck
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Rat Plasma ◽  
Human Platelets ◽  
High Concentrations ◽  
Low Sensitivity ◽  
Von Willebrand ◽  
Induced Aggregation ◽  
Willebrand Factor ◽  
Rat Platelets

SummaryRat platelets do not respond to ristocetin in their own plasma nor do they aggregate in the presence of bovine or porcine factor VIII von Willebrand factor (F VIII R:WF) or human F VIII R:WF in presence of ristocetin. However, rat plasma supports ristocetin induced aggregation of washed human platelets. In this study we report on purification of rat F VIII R:WF from cryoprecipitate. Similarly to porcine or bovine material, purified rat F VIII R:WF induced aggregation of human washed fixed platelets. This effect was enhanced by addition of ristocetin and was not modified by addition of albumin. Rat washed platelets were aggregated by ristocetin in the presence of rat or human F VIII R:WF provided that high concentrations of ristocetin are added in a system essentially free of extraneous proteins. Increasing concentrations of albumin dramatically reduced the ability of ristocetin to aggregate rat platelets while human platelet aggregation by human or rat F VIII R:WF was only moderately affected.These studies show that rat F VIII R:WF can interact with rat and human platelets. The lack of response of rat platelets to ristocetin in their own plasma is most likely due to a low sensitivity of rat platelets to this drug and to an inhibitory activity of plasma proteins on this reaction.

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